The objective of this proposal is to bioengineer a mutant variant of prourokinase (proUK) with improved therapeutic properties for treatment of acute myocardial infarction(AMI). ProUK is unique among thrombolytic agents in that it is stable as an inert zymogen in blood plasma before activation and conversion to urokinase (UK) at the site of a fibrin blot clot, at least under physiological concentrations. There are several discrete properties of proUK which we have targeted for improvement: (a) increased fibrin specificity, (b) increased resistance to natural inhibitors such as plasminogen activator inhibitor (PAI), (c) reduced nonspecific systemic bleeding, (d) increased half life, and (e) single bolus administration. Bioengineered versions of tissue plasminogen activator (tPA) which have been introduced into the clinic recently, such as Tenecteplase and Reteplase, do not adequately fulfill these ideal properties, so there is ample opportunity to develop other therapeutics with superior qualities, such as bioengineered proUK. The bioengineered variants of proUK will be expressed in E. coli and refolded and purified from insoluble inclusion bodies. We have already refolded and purified wild type proUK from inclusion bodies, and demonstrated that upon activation to UK, the kinetic parameters related to fibrinolysis show excellent agreement with published values. Our company, Proteomtech, is a recognized leader in the area of protein refolding technology from inclusion bodies and has already successfully refolded more than 200 proteins, some at industrial scale. Published studies have established that nonglycosylated wild type proUK and UK expressed in E. coli have superior, quicker, fibrinolytic properties when compared to glycosylated versions expressed in eukaryotic expression systems; the challenge is to increase proUK half life and stability in vivo for the unglycosylated proUK. Our long-term objective is to develop the bioengineered variant for clinical application to AMI with the possibility of extending to other conditions including stroke, peripheral arterial occlusion, and deep vein thrombosis. No preclinical animal studies or human studies are included in this proposal.